Genome-wide location analysis by pull down of in vivo biotinylated transcription factors.
Genome-wide location analysis by pull down of in vivo biotinylated transcription factors.
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DOI：
10.1002/0471142727.mb2120s92
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发表时间：
2010-10
影响因子：
--
通讯作者：
Pu, William T
中科院分区：
文献类型：
--
作者：
He, Aibin;Pu, William T
最近发展的方法，全基因组识别的转录因子结合位点的染色质免疫沉淀（ChIP）导致新的见解转录调控和更好地理解个别转录因子的功能。染色质免疫沉淀需要针对感兴趣的转录调节因子的高度特异性抗体，并且合适抗体的可用性是该方法更广泛应用的重大障碍。这种限制可以通过用短的生物表位标记感兴趣的转录调节子来规避，所述短的生物表位被E.大肠杆菌酶BirA。然后，在严格条件下，生物素化的转录因子可以选择性地拉下链霉亲和素珠。该单元提供了一个详细的协议，通过链霉亲和素下拉，然后高通量测序（bioChIP-seq）在体内生物素化的转录因子的全基因组定位分析。
Recent development of methods for genome-wide identification of transcription factor binding sites by chromatin immunoprecipitation (ChIP) has led to novel insights into transcriptional regulation and greater understanding of the function of individual transcription factors. Chromatin immunoprecipitation requires highly specific antibody against the transcriptional regulator of interest, and availability of suitable antibodies is a significant impediment to broader application of this approach. This limitation can be circumvented by tagging the transcriptional regulator of interest with a short bio epitope, which is specifically biotinylated by the E. coli enzyme BirA. The biotinylated transcription factor can then be selectively pulled down on streptavidin beads under stringent conditions. This unit provides a detailed protocol for genome-wide location analysis of in vivo biotinylated transcription factors by streptavidin pull-down followed by high throughput sequencing (bioChIP-seq).